Mycobacteria culture medium and method including mycobacteria of mycobacterium tuberculosis complex

Abstract

The present invention relates to a novel mycobacteria culture medium, particularly for mycobacteria of the Mycobacterium tuberculosis complex, that significantly reduces the culture isolation time and thus the time for diagnosing the mycobacteria, particularly those for tuberculosis. A culture medium according to the invention contains defibrinated blood, lecithin, and decomplemented fetal calf serum. The present invention also relates to a culture method and to a method for identifying mycobacteria, particularly bacteria of the Mycobacterium tuberculosis complex. The present invention also relates to a novel decontamination method through chlorhexidine treatment of biological samples in an isolation and mycobacteria culture medium, and to a method for determining by phenotype the sensitivity of mycobacteria to antibiotics by means of a solid culture medium according to the invention. Finally, the present invention also relates to a method of identification in liquid phase by mass spectrometry which reduces time for diagnosing mycobacteriosis, particularly for tuberculosis.

The invention claimed is: 
     
       1. A culture medium for mycobacteria, comprising:
 growth factors for mycobacteria, 
 antibiotics without activity against mycobacteria,
 lecithin in a proportion by weight of from 0.1 to 5%, and 
 
 defibrinated blood in a proportion by volume of from 2.5 to 15%, and 
 decomplemented fetal calf serum in a proportion by volume of from 2.5 to 25%. 
 
     
     
       2. The culture medium for mycobacteria as claimed in  claim 1 , wherein:
 the lecithin is egg yolk lecithin, and 
 the blood is defibrinated rabbit or sheep blood. 
 
     
     
       3. The culture medium for mycobacteria as claimed in  claim 1 , wherein said culture medium comprises a basic culture medium for mycobacteria comprising, besides distilled water, the following components: ammonium sulfate, magnesium sulfate, copper sulfate, zinc sulfate, sodium citrate, ferric ammonium citrate, sodium chloride, calcium chloride, monopotassium phosphate, disodium phosphate, L-glutamic acid, biotin and pyridoxine. 
     
     
       4. The culture medium for mycobacteria as claimed in  claim 1 , wherein said culture medium is a liquid culture medium. 
     
     
       5. The culture medium for mycobacteria as claimed in  claim 4 , wherein said culture medium comprises a basic culture medium which is a Middlebrook liquid medium of reference 7H9 and the following additional growth factors for mycobacteria: casein hydrolyzate, glycerol, polysorbate, hemin, lactic acid, biotin, polyoxyethylene stearate, bovine serum albumin, dextrose and supplement H. 
     
     
       6. The culture medium for mycobacteria as claimed in  claim 1 , wherein said culture medium is a solid culture medium containing a gelling agent. 
     
     
       7. The culture medium for mycobacteria as claimed in  claim 6 , wherein said culture medium is a solid medium of the Middlebrook type of reference 7H10, with addition of said gelling agent and the following additional growth factors for mycobacteria: oleic acid, bovine albumin, dextrose, catalase and glycerol. 
     
     
       8. The culture medium for mycobacteria as claimed in  claim 1 , wherein lecithin is contained in a proportion by weight from 0.5 to 1%. 
     
     
       9. The culture medium for mycobacteria as claimed in  claim 8 , wherein said culture medium is a solid culture medium containing a gelling agent selected from agars in a proportion by weight from 0.5 to 5%. 
     
     
       10. The culture medium for mycobacteria as claimed in  claim 1 , wherein the blood is contained in a proportion by volume from 5 to 10%. 
     
     
       11. The culture medium for mycobacteria as claimed in  claim 1 , wherein decomplemented fetal calf serum is contained in a proportion by volume from 10 to 20%.